Food quality of mixed bacteria–algae diets for Daphnia magna
Heike M. Freese•Dominik Martin-Creuzburg
Abstract Bacteria can comprise a large fraction of seston in aquatic ecosystems and can therefore signif- icantly contribute to diets of filter-feeding zooplank- ton. To assess the effect of three heterotrophic bacteria (Flavobacterium sp., Pseudomonas sp. and Esche- richia coli) on survival, growth and egg production of juvenile Daphnia magna during six-day growth experiments, five ratios of bacteria Scenedesmus obliquusmixtures were fed. Potential growth-limiting effects mediated by essential biochemicals were assessed upon supplementation of pure bacterial diets with a sterol (cholesterol) or a polyunsaturated fatty acid (EPA). Pure bacterial diets always had detrimen- tal effects on Daphnia. However, cholesterol supple- mentation of Flavobacterium sp. enhanced growth
rates of Daphnia. Diets containing Pseudomonas impairedDaphniagrowth even at low dietary propor- tions (20%), indicating their toxicity. In contrast, Daphniagrew at relative high dietary proportions of Flavobacteriumsp. andE. coli(80 50%). In fact, diets containing small proportions of these heterotrophic bacteria (Flavobacterium B50%, E. coli 20%) even significantly increased Daphnia growth rates com- pared to pure algal diets, indicating a nutritional upgrading by these bacteria. Our results suggest that the relative contribution of bacteria and phytoplankton to total dietary carbon as well as their phylogenetic composition strongly influence Daphnia fitness and potentially other filter-feeding zooplankton under field conditions.
Keywords Flavobacteriumsp.Pseudomonassp.
Escherichia coliPolyunsaturated fatty acids SterolsScenedesmus
Introduction
Aquatic systems are characterized by a complex food web, in which organic matter is transferred across different trophic levels. Cladocerans of the genus Daphniaoften dominate the zooplankton in standing freshwater systems, and thus provide a crucial link between primary and secondary production (Peters &
de Bernardi, 1987). As non-selective filter-feeders, Daphniado not only graze on phytoplankton but also
Guest editors: Marina Manca & Piet Spaak / Cladocera:
Proceedings of the 9th International Symposium on Cladocera H. M. Freese
Department of Biology, Microbial Ecology, University of Konstanz, 78457 Konstanz, Germany H. M. Freese (&)
Microbial Ecology and Diversity Research, Leibniz Institut DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstraße 7B, 38124 Braunschweig, Germany
e mail: heike.freese@dsmz.de D. Martin Creuzburg
Limnological Institute, University of Konstanz, Mainaustrasse 252, 78464 Konstanz, Germany
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-214668
on protozoa, bacteria and detritus (Ju¨rgens,1994; Cole et al.,2006). Heterotrophic bacteria often constitute a substantial part of the suspended particulate organic matter and have a key function by channelling organic carbon via incorporation into the food web (Azam et al., 1983; Biddanda et al., 2001). The aquatic bacterial communities consist of many different populations from different phyla (e.g. Rappe &
Giovannoni,2003). Freshwater ecosystems are mainly dominated by b-proteobacteria, actinobacteria and bacteroidetes, but a-proteobacteria, verrucomicrobia or planctomycetes can also be abundant (cf. Zwart et al.,2002,2003; Newton et al.,2011).Daphniacan shape these microbial communities either by grazing on bacterivorous protozoans or by direct consumption of bacteria (Langenheder & Ju¨rgens, 2001; Degans et al.,2002; Pernthaler et al.,2004).
Although bacteria can be efficiently consumed by Daphnia (Gophen & Geller, 1984; Brendelberger, 1991), their importance forDaphnianutrition has been considered only recently. Analysis of stable isotope patterns and fatty acid biomarkers revealed that heterotrophic bacteria can significantly contribute to the diet ofDaphniaspecies (Karlsson et al.,2003; Perga et al., 2006; Taipale et al., 2008, 2009). Bacteria generally have higher phosphorus to carbon (P:C) ratios than algae (Vadstein,2000), and thus may sustain the high P demand ofDaphnia(Andersen & Hessen,1991;
Vrede et al., 1999; Hessen et al., 2002). Besides P, Daphnia also require essential biochemicals, like polyunsaturated fatty acids (PUFAs) and sterols (Brett
& Mu¨ller-Navarra, 1997; Martin-Creuzburg et al., 2005, 2009), which are important membrane compo- nents and serve as precursors for a number of bioactive molecules (Grieneisen, 1994; Harrison et al., 1997;
Desvilettes et al.,1997; Martin-Creuzburg et al.,2007).
Only few bacteria (methanotrophic bacteria, MOB) are known to produce sterols (e.g. Schouten et al., 2000;
Volkman, 2003), but stable isotope analysis indicate that these bacteria may significantly contribute to Daphniadiet (Taipale et al.,2007,2008). In addition, a MOB has been reported to enhance the reproduction ofDaphnia, but the growth ofDaphniaitself was not enhanced (Taipale et al.,2012). Only MOB parallel fed with limited quantities of phytoplankton partially supportDaphniagrowth (Deines & Fink,2011). Since sterol supplementation of bacterial diets (including a MOB) enhanced growth of D. magna(Martin-Creuz- burg et al., 2011), the quality of bacteria as sole food
source forDaphniais likely low due to the absence of sterols in bacteria. Long chain PUFAs occur also not commonly in bacteria, although some, mostly marine psychrophilic bacteria did contain PUFAs (Russell &
Nichols,1999; Okuyama et al.,2007). Consequently, Daphniahave been shown to be limited simultaneously by the absence of sterols and long chain PUFAs when feeding on cyanobacterial diet (Martin-Creuzburg et al., 2008,2009). Thus,Daphniaare likely to be additionally restricted by the absence of these biochemicals when feeding on heterotrophic bacterial diets.
In natural environments, bacteria are unlikely the sole food source forDaphniaand instead are ingested along with phytoplankton species. The bacteria/phyto- plankton proportions vary strongly among lakes and within lakes depending on nutrient state and season (0.02 16 bacterial-C/phytotplakton C; cf. Simon et al., 1992; Hessen et al., 2003). Here, to investigate the response of survival, growth and egg production of D. magna to differences in food quality of mixed bacteria-phytoplankton diets, we conducted standard- ized growth experiments with juvenile animals feeding on different bacteria provided either as sole food source or in combination with the green alga Scenedesmus obliquus. By experimentally increasing the proportion of different bacteria in the diet, we determined the bacteria to phytoplankton ratio at whichDaphniawere negatively affected indicating the effect of declined food quality. We hypothesized that higher proportions of heterotrophic bacteria in the food suspension will restrict growth and egg production ofDaphniabecause of a limitation by sterols and PUFAs and that low proportions will either not affect or even benefit the animals. In addition, to assess the significance of a dietary sterol or PUFA deficiency caused by increasing the proportion of bacterial carbon, bacteria were supplemented either with cholesterol, the predominant animal sterol, or eicosapentaenoic acid (EPA), a long chain PUFA known to be of particular importance for Daphniagrowth and reproduction.
Materials and methods
Cultivation of food organisms and preparation of food suspensions
Growth experiments were conducted with three strains of heterotrophic bacteria, i.e. Pseudomonas sp. DD1
(NCBI: HQ113379) and Flavobacterium sp. DD5b (NCBI: HQ113381), both representing typical pelagic bacteria (Glo¨ckner et al., 2000; Van der Gucht et al., 2005; Pearce et al., 2005), and E. coli (wild-type strain), which is regularly found in aquatic ecosystems (LaLiberte & Grimes, 1982 and cited references;
Hamelin et al.,2007). The bacterial strains were grown in mineral medium as described in Martin-Creuzburg et al. (2011), but with 20 mM glucose and trace element solution SL12a (2 ml l-1) which was slightly modified with 1.1 g l-1 FeCl24H2O, 0.07 g l-1ZnCl2
and 0.1 g l-1 MnCl2 from SL 12 (Overmann et al., 1992). Bacteria were grown at 20°C and harvested daily in the late exponential and early stationary growth phase. Cells were centrifuged (10 min, 5,000g, 15°C), and resuspended in sterile-filtered and autoclaved Lake Constance water. Aggregates were dissolved by vor- texing and short sonication. In order to add defined numbers (carbon concentrations) of bacteria to the growth experiments (Table1), cell numbers were determined in a Helber counting chamber with a Zeiss Axiophot microscope. Carbon concentrations of defined bacterial cell numbers (i.e. bacterial carbon content) were estimated before the start of the exper- iment to adjust the carbon concentrations and were repeated during the experiment.
The green alga S. obliquus (SAG 276-3a), which contains sterols, but is deficient in C-20 PUFAs and thus of intermediate food quality, was used as food for stock cultures ofD. magnaand as a reference food in the growth experiments. It was grown in semi- continuous batch cultures as described in Martin-
Creuzburg et al. (2005) and harvested in the late exponential growth phase. Carbon contents of the autotrophic food suspensions were estimated from photometric light extinctions (800 nm) and from previously determined carbon-extinction equations.
Daphniagrowth experiments
Stock cultures of D. magna (originally isolated by Lampert, 1991) were raised in filtered lake water (0.2- lm pore-sized membrane filter) containing saturating concentrations ofS. obliquus. Growth experiments were carried out with third-clutch neonates (born±6 h) at 20°C in glass beakers filled with 200 ml of filtered lake water (\0.2lm). Each treatment consisted of three replicates with sixD. magnaper beaker. Animals were transferred daily into new beakers with freshly prepared food suspensions over a period of 6 days, after which eggs were produced and the effect of different food qualities were pronounced.
Daphnia magna were raised on Pseudomonas sp.
DD1,Flavobacterium sp. DD5b, or E. coli either as sole food source (100:0%, i.e. 2 mg C l-1) or in different combination withS. obliquus(bacteria:phy- toplankton carbon proportions: 80:20%, 50:50%, 20:80%, 0:100%; Table 1). The total carbon concen- tration in all treatments was 2 mg C l-1. In addition, pure bacterial food suspensions were supplemented with either 50 ll of control liposomes (no sterols, no PUFAs), 50ll cholesterol-containing liposomes, or 50ll EPA-containing liposomes per beaker. Lipo- some stock suspensions were prepared as described in
Table 1 Experimental setup and composition of food suspension Treatment Supplementation
of bacteria with
Bacterial carbon (mg C l-1)
Abundance of Flavobacteriumsp.
DD5b (ml-1)
Abundance of Pseudomonassp.
DD1 (ml-1)
Abundance of E. coli(ml-1)
Starving Nothing 0 0 0 0
Scenedesmus S. obliquus 0 0 0 0
20/80 S. obliquus 0.4 49106 49106 4.49106
50/50 S. obliquus 1 109106 109106 11.19106
80/20 S. obliquus 1.6 169106 169106 17.89106
Bacteria Nothing 2 209106 209106 22.29106
?Liposoms Control liposomes 2 209106 209106 22.29106
?Cholesterol Cholesterol containing liposomes
2 209106 209106 22.29106
?EPA EPA containing liposomes 2 209106 209106 22.29106
Martin-Creuzburg et al. (2008). The experiment was completed by a concomitant starvation treatment.
To estimate somatic growth rates of D. magna, subsamples of the experimental animals were taken at the beginning and at the end of the experiment, dried for 24 h, and weighed on an electronic balance (Mettler Toledo XP2U; ±0.1 lg). Somatic growth rates (g) were determined as the increase in dry mass from day 0 (M0) to day 6 (M6) of the experimental period (t=6 days) using the equation:g =(lnM6 lnM0) t-1. Egg production was estimated by counting the eggs in the brood chambers of the animals at the end of the experiment.
Biochemical analyzes of food suspensions
For analysis of fatty acids and sterols, at least 59109 cells were harvested by centrifugation, washed, freeze-dried, and stored at-80°C at two time points.
Total lipids were extracted thrice from freeze-dried samples with dichloromethane/methanol (2:1, v/v) and the pooled cell-free extracts were evaporated to dryness with nitrogen. Lipid extracts were transeste- rified with 3 mol l-1methanolic HCl (60°C, 15 min) for analysis of fatty acids, or saponified with 0.2 mol l-1methanolic KOH (70°C, 1 h) for analysis of sterols. Subsequently, fatty acid methyl esters (FAMEs) were extracted three times with 2 ml iso- hexane; the neutral lipids were partitioned into iso- hexane:diethyl ether (9:1, v/v). The lipid-containing fraction was evaporated to dryness under nitrogen and resuspended in a volume of 10 20ll iso-hexane.
Lipids were analyzed by gas chromatography on a HP 6890 GC equipped with a flame ionization detector (FID) and a DB-225 (J&W Scientific) capillary column to analyze FAMEs or with a HP-5 (Agilent Technologies) capillary column to analyze sterols.
Details of GC configurations are given elsewhere (for fatty acids, Martin-Creuzburg et al. (2010); for sterols, Martin-Creuzburg et al. (2009)). Lipids were quanti- fied (FID) by comparison to internal standards (C23:0 ME, 5a-cholestan) of known concentrations using multipoint standard calibration curves previously established for each compound (Sigma-Aldrich). The few non-purchasable lipid compounds were quantified using calibration curves of structurally related lipids with similar retention times. Lipids were identified by their retention times and their mass spectra, which were recorded with a gas chromatograph-mass
spectrometer (Agilent Technologies, 7890A GC, 5975C inert MSD) equipped with a fused-silica capillary column (DB-225MS, J&W for FAMEs;
DB-5MS, Agilent for sterols). Sterol samples were analyzed in their free form and as their trimethylsilyl derivatives. Spectra were recorded between 50 and 600 amu in the EI ionization mode. The limit of quantitation was*20 ng for fatty acids or sterols. The absolute amount of each lipid was related to the particulate organic carbon (POC). At three times during the experiment, carbon, nitrogen (N) and sulphur (S) were determined in duplicates from bacterial and algal suspensions concentrated in tin capsules for liquid samples using an elemental analyser (EuroEA3000, HEKAtech GmbH, Ger- many). For determination of particulate phosphorus, two aliquots per food suspension were collected on acid-rinsed polysulfone filters (HT-200; Pall) and digested with a solution of 10% potassium peroxodi- sulfate and 1.5% sodium hydroxide for 60 min at 121°C; soluble reactive phosphorus was determined using the molybdate-ascorbic acid method (Greenberg et al.1985).
Statistical analysis
Somatic growth rates and egg production ofDaphnia magna were analyzed using one-way analyses of variance (ANOVA) and post hoc tests (Tukey’s HSD or Dunnett’s T3 if variances were not equal (E. coli)).
Treatments in which only one or none animal per beaker survived were excluded from the ANOVAs.
Raw data met the assumptions for ANOVA. Statistical analyses were carried out using the General Linear Model module of SPSS 11 (SPSS Inc.).
Results
Characteristics of food sources
The three bacterial strains used for diet mixing experiments hardly differed in their C, N, and S-content per cell (Table2). The phosphorus content per cell was lowest in E. coli and highest in Flavo- bacterium sp. Compared to S. obliquus, the bacteria were characterized by lower C:N (33 45%) and C:P ratios (22 46%), indicating a higher N and P content of the bacteria (Table2).
The fatty acid (FA) composition of the bacterial strains was dominated by saturated and monounsatu- rated fatty acids (Table3). The fatty acid profiles of the c-proteobacteria E. coli and Pseudomonas sp. were similar except for the saturated fatty acid 19:0 which only occurred in E. coli, a higher proportion of palmitoleic (16:1n-7) acid inPseudomonassp., and a higher proportion of cyclopropanoic acid (17:0D) in E. coli. The FA profile ofFlavobacteriumsp. was more diverse than the profiles of the other two bacteria and contained also high amounts of branched-chain and hydroxy-fatty acids. PUFAs and sterols could not be detected in any of the bacterial strains. In contrast, S. obliquus contained high amounts of 18:2n-6 and 18:3n-3, but no PUFAs with more than 18 carbon atoms. Chondrillasterol (IUPAC name: (22E)-5a- poriferasta-7,22-dien-3b-ol; mean±SD: 58.3±3.8%), fungisterol (5a-ergost-7-en-3b-ol; 19.4 ±1.6%), 22-dihydrochondrillasterol (5a-poriferast-7-en-3b-ol;
9.7 ±0.9%), and schottenol (5a-stigmast-7-en-3b-ol;
12.5 ±1.1%) were the principal sterols found in the green alga.
The supplemented liposomes did not differ in their palmitic acid (16:0) and oleic acid (18:1n-9) content, which both are components of the phospholipids used to prepare the liposomes (Martin-Creuzburg et al., 2008). Liposomes prepared either in the presence of EPA or in the presence of cholesterol contained 14.5 ±1.9 lg EPA or 11.2±1.1 lg cholesterol per 50 ll of liposome stock suspension, respectively.
Survival ofD. magna
On a pure S. obliquus diet, all Daphnia survived the experimental period. Without food, about 40% of the animals survived (Fig.1). On pure bacterial diets,
survival differed depending on the bacterial strain used.
In general, survival of D. magna was highest on Flavobacterium sp. (*60%), intermediate on E. coli (*15%), and lowest onPseudomonassp. (0%). When provided in combination withS. obliquus, survival of D. magna decreased with increasing proportions of bacteria in the food suspension (Fig.1). This decrease in survival was most pronounced with Pseudomonas sp.; none of the animals survived the experimental period when C50% of the available carbon was provided asPseudomonassp. In contrast, survival on diets containing increasing proportions ofFlavobacte- rium sp. or E. coliwas not reduced until 80% of the available carbon was bacterial carbon (Fig.1). Cho- lesterol supplementation of a pureFlavobacteriumsp.
diet increased survival of D. magna, but cholesterol supplementation did not affect survival on the c-proteobacteria. In contrast, survival on thec-proteo- bacteria increased upon EPA supplementation (Fig.1).
Somatic growth rates and egg production ofD. magna
Somatic growth rates ofD. magnawere significantly affected by increasing the proportion of bacteria in their diet. Exchanging 20% of the available carbon with Flavobacterium sp. or E. coli slightly but significantly increased somatic growth rates as com- pared to those obtained on a pure S. obliquus diet (Fig.2a, c; Tukey’s HSD (a) or Dunnett’s T3 (c), P\0.05 following ANOVA: F4,10 =1506 (a), F3,8=220 (c), bothP\0.001). In general, however, somatic growth rates decreased with increasing proportions of bacterial dietary carbon. With Flavobacterium sp. and with E. coli, this decrease was significant at proportions C80% and with
Table 2 Elemental composition and proportion of bacterial strains in comparison to the green algaeScenedesmus obliquus Flavobacterium sp. DD5b Pseudomonassp. DD1 E. coli S. obliquus
fg C/cell 90.6±18.4 81.9±24.1 77.7±9.9
fg N/cell 21.2±4.3 21.9±5.2 22.9±2.6
fg P/cell 4.4±0.1 3.0±0.02 2.3±0.04
fg S/cell 3.7±1.0 3.3±1.0 3.4±0.9
C:N (mol:mol) 4.9 4.2 4.0 7.3
C:P (mol:mol) 64 82 92 117
Data represent means of three replicates (for phosphate one) over time from which each two subsamples were measured±standard deviation
Pseudomonas sp. somatic growth rates significantly decreased already with 20% of bacterial carbon (ANOVA: F1,4 =644, P\0.001; Fig.2). Growth ofDaphniadiffered significantly in dependence of the fed bacterial phylotype, e.g. at 20% bacterial carbon, Pseudomonassp. fedDaphniagrew significantly less than Flavobacterium sp. or E. coli fed Daphnia (Tukey’s HSD, P\0.05 following ANOVA:
F2,6 =1041) and at 50%, E. coli fedDaphnia grew significantly less than Flavobacterium sp. fedDaph- nia carbon (ANOVA: F1,4 =27, P\0.01). When provided as sole food source, all bacteria were highly detrimental forD. magna. On a purePseudomonassp.
diet, none of the animals survived the experimental
period and on a pureE. colidiet, only animals of one replicate barely survived so that somatic growth rates could not be determined. Growth rates obtained on a pure Flavobacterium sp. diet were low, but signifi- cantly increased upon cholesterol supplementation up to growth rates obtained on a pure S. obliquus diet (Tukey’s HSD, P\0.05 following ANOVA:
F4,10= 255; Fig. 2d). Supplementation ofFlavobac- teriumsp. with control liposomes or EPA-containing liposomes did not improve somatic growth rates.
Liposome supplementation of E. coli slightly improved survival and thus somatic growth rates could be determined. The obtained growth rates, however, were low and not affected by cholesterol or
Table 3 Fatty acid content of the three bacteriaFlavobacterium sp.,Pseudomonassp. andE. coli, and of the green algaScene desmus obliquus
Flavobacterium (% of total FA)
Pseudomonas (% of total FA)
E. coli
(% of total FA)
S. obliquus (% of total FA)
14:0 1.39±0.14 0.46±0.12 0.91±0.19 0.98±0.11
15:0 9.33±0.16 n.d. n.d. n.d.
i15:0 10.09±0.17 n.d. n.d. n.d.
a15:0 1.41±0.00 n.d. n.d. n.d.
2 OH 15:0 4.88±0.06 n.d. n.d. n.d.
3 OH 15:0 2.06±0.08 n.d. n.d. n.d.
15:1n[5 2.16±0.10 n.d. n.d. n.d.
15:1n 5 2.12±0.09 n.d. n.d. n.d.
16:0 13.14±0.53 32.06±0.48 28.09±0.06 22.62±0.82
i16:0 4.80±0.09 n.d. n.d. n.d.
3 OH i16:0 1.88±0.05 n.d. n.d. n.d.
3 OH a16:0 3.07±0.08 n.d. n.d. n.d.
3 OH 17:0 2.84±0.10 n.d. n.d. n.d.
16:1n[7 1.16±0.05 n.d. n.d. n.d.
16:1n 7 28.51±0.39 36.68±0.61 1.55±0.04 0.34±0.09
16:1n\7 0.76±0.00 n.d. 0.41±0.04 n.d.
i17:0 1.50±0.01 n.d. n.d. n.d.
17:0D 2.04±0.26 4.70±0.03 24.77±0.01 n.d.
17:1n 7 3.34±0.05 n.d. n.d. n.d.
18:0 2.56±0.48 2.03±0.02 1.79±0.11 3.76±0.27
18:1n 9/n 12 0.96±0.06 24.06±0.04 27.84±0.64 26.5±0.51
19:0D n.d. n.d. 14.65±0.27 n.d.
18:1n 7 n.d. n.d. n.d. 0.41±0.13
18:2n 6 n.d. n.d. n.d. 11.63±0.23
18:3n 3 n.d. n.d. n.d. 30.60±0.97
18:4n 3 n.d. n.d. n.d. 3.16±0.19
Data represent means of two (bacteria) or three (alga) replicates over time±standard deviation (n.d.not detectable, i.e.\20 ng)
EPA supplementation. Liposome supplementation could also not improve somatic growth ofD. magna on a purePseudomonassp. diet (Fig.2).
As observed for somatic growth, the detrimental effect of bacterial carbon onDaphniaegg production increased with decreasing proportions of dietary S. obliquus(Fig. 3). Animals raised on a diet consist- ing ofC80%Flavobacteriumsp., did not produce eggs within the experimental period and egg production was significantly reduced on a diet consisting ofC50%
E. coli(Tukey’s HSD, P\0.05 following ANOVA:
F2,6=20). Animals exposed toPseudomonassp. did not produce eggs, even at the lowest dietary concen- tration. Animals raised on pure Flavobacterium sp.
produced eggs upon cholesterol supplementation but less than produced on a pureS. obliquusdiet.
Discussion
It has been recognized that heterotrophic bacteria are of poor food quality for Daphnia(Martin-Creuzburg
D G
E H
F I
Fig. 1 Survival of juvenile D. magna exposed to different bacteria/Scenedesmus obliquusmixtures (aFlavobacteriumsp., b Pseudomonas sp., and c E. coli) as well as to bacteria supplemented with cholesterol or EPA containing liposomes or control liposomes (Lipos) without added EPA or cholesterol
(d Flavobacterium sp., e Pseudomonassp., andf E. coli) in comparison to starved animals. Data were calculated from the numbers of individuals which survived the experimental period of 6 days (means ofn 3 jars)
et al.,2011; Wenzel et al.,2012; Taipale et al.,2012).
In a previous study, we have shown that this poor food quality is partially due to a dietary deficiency in sterols, as indicated by a growth-enhancing effect upon sterol supplementation of different bacterial diets (Martin-Creuzburg et al., 2011). However, bacteria are not only characterized by a deficiency in
sterols, they usually are also deficient in PUFAs (Russell & Nichols, 1999; Okuyama et al., 2007), suggesting a co-limitation by sterols and PUFAs as has been shown for cyanobacterial diets (Martin-Creuz- burg et al., 2009). Moreover, a number of bacterial strains isolated from aquatic habitats have been shown to produce toxic secondary metabolites that are active
D G
E H
F I
Fig. 2 Somatic growth rates of juvenileD. magnaon different diets in comparison to starved animals. Daphnia were fed different bacteria/Scenedesmus obliquusmixtures (aFlavobac teriumsp.,bPseudomonassp., andcE. coli) as well as bacteria supplemented with cholesterol or EPA containing liposomes or control liposomes (Lipos) without added EPA or cholesterol (d Flavobacterium sp., e Pseudomonas sp., and f E. coli).
Growth rates of animals fed 100%E. coliwere excluded since only few animals of one replicate survived. The horizontal gray bar indicates growth rates ofD. magnafedS. obliquus. Data are means of three replicates per treatment;error barsindicate SD.
Bars labelled with the same letters are not significantly different (Tukey’s HSD,P\0.05 following ANOVA)
towards protozoans and metazoan grazers, among themD. magna(Matz & Kjelleberg,2005; Matz et al., 2008; Deines et al.,2009). High bacterial toxicity was also observed in our previous study, in which D.
magnawere exposed to aHydrogenophagasp. strain or a Pseudomonas sp. strain, which were previously isolated from the digestive tract ofD. magna(Martin- Creuzburg et al., 2011). The high toxicity of this
Pseudomonassp. strain was corroborated in this study.
When provided in combination with S. obliquus, growth and egg production ofD. magnawere signif- icantly impaired even at low dietary concentrations of Pseudomonas sp. (20% of dietary carbon). When Pseudomonas sp. was provided in higher concentra- tions, none of the animals survived the experimental period of 6 days. Pseudomonas spp. are known to
D G
E H
F I
Fig. 3 Egg production of D. magna on different diets in comparison to starved animals. Daphnia were fed different bacteria/Scenedesmus obliquusmixtures (aFlavobacteriumsp., b Pseudomonas sp., and c E. coli) as well as bacteria supplemented with cholesterol or EPA containing liposomes or control liposomes (Lipos) without added EPA or cholesterol
(dFlavobacteriumsp.,ePseudomonassp., andfE. coli). The horizontal gray bar indicates number of eggs ofD. magnafedS.
obliquus. Data are means of three replicates per treatment;error bars indicate SD. Bars labelled with the same letters are not significantly different (Tukey’s HSD, P\0.05 following ANOVA)
produce a variety of toxic secondary metabolites (Gross & Loper, 2009), suggesting that the negative effects of the isolatedPseudomonas sp. strain on the performance ofD. magnawe observed here and in our previous study were also caused by toxic secondary metabolites. Numerous Pseudomonas species/strains have been shown to act as pathogens in a wide range of invertebrates (Padmanabhan et al., 2005; e.g. Hilbi et al.,2007); detrimental effects onDaphniahave been reported for P. aeruginosa and P. entomophila (Le Coadic et al., 2012). However, Wenzel et al. (2012) reported thatDaphniacan survive on diets containing high proportions of another Pseudomonas strain, indicating thatPseudomonasstrains are not necessar- ily detrimental to consumers.
A somewhat lower mortality was observed on diets containing E. coli. AlthoughE. coliis mostly classi- fied as commensal, the potential of some strains to cause diseases in humans and other mammals has long been recognized. Less is known about pathogenicity of E. colifor invertebrates. However,E. colistrains were found to act as opportunistic pathogens in stressed and immunocompromised invertebrates, i.e. in old indi- viduals and in individuals exposed to other pathogens [Millet & Ewbank,2004(nematode); Broderick et al., 2006 (gypsy moth larvae)]. Since higher concentra- tions ofE. colithanPseudomonassp. were required to cause the death of Daphnia, one may speculate that high amounts of a low quality food which likely reduced fitness of the animals increased Daphnia susceptibility to the pathogens. On the other hand, most E. coli strains from aquatic environments are non-pathogenic (e.g. Hamelin et al., 2007). The detrimental effect of higher proportions of E. coli may not be caused by a possible pathogenicity (or toxicity) but by a more pronounced response of Daphnia to their restricted food quality, since the effect of food quality increase with food quantity (Sterner,1997).
The mortality ofD. magnaraised on diets contain- ing Flavobacterium sp. was far less pronounced and presumably caused by nutritional challenges rather than toxicity. This at least was suggested by the growth-enhancing effect of sterol supplementation, indicating a sterol limitation of D. magna. In fact, somatic growth rates of D. magna on a pureFlavo- bacterium sp. diet increased upon sterol supplemen- tation up to the growth rates obtained on a pure S. obliquusdiet, showing that the absence of sterols is
the major food quality constraint daphnids are con- fronted with while feeding on this bacterium. We propose that the negative effects associated with a dietary deficiency in sterols are on the tested c-proteobacteriaPseudomonassp. andE. colimasked by the toxicity of these bacteria.
It has been shown that daphnids feeding on cyanobacteria are simultaneously limited by sterols and PUFAs (Martin-Creuzburg et al.,2009; Sperfeld et al.,2012). In a previous study, we did not find clear evidence for such a co-limitation of Daphnia while feeding on heterotrophic bacteria (Martin-Creuzburg et al., 2011). In this study, supplementation of heterotrophic bacteria with EPA did not increase somatic growth rates or egg production ofD. magna, suggesting that the absence of dietary PUFAs did not constrain the performance of the animals. This is somehow supported by the finding that somatic growth rates ofD. magnaon a pureFlavobacteriumsp. diet, which contained neither EPA nor other PUFAs, increased upon sterol supplementation up to the level obtained on a pure algal (S. obliquus) diet. However, dietary PUFAs are indispensable for proper growth and reproduction ofDaphnia, as has been shown by numerous studies, and thus it is rather unlikely that the performance of animals feeding on heterotrophic bacteria is not affected by the absence of dietary PUFAs. One might argue that the experimental design we used here, i.e. short-term growth experiments, are unsuitable to detect potential consequences associated with a dietary PUFA deficiency, because the animals may still rely on maternal PUFA reserves. However, the maternal PUFA supply was presumably low, because the animals were pre-raised onS. obliquus, a green alga deficient in long chain PUFAs. Although, these PUFAs can be produced and retained from shorter algal PUFAs byDaphnia(Kainz et al., 2004, Taipale et al.,2011), previous studies have repeatedly shown that even short-term feeding on PUFA deficient diets results in a limitation by PUFAs, provided that at least small amounts of dietary sterols are available (Martin-Creuzburg et al.,2009; Sperfeld et al.,2012).
In our previous study, however, simultaneous supple- mentation ofFlavobacteriumsp. with cholesterol and EPA did also not reveal clear evidence for a PUFA limitation once sterol requirements were met (Martin- Creuzburg et al.,2011). Overall, we did not find clear evidence for a limitation by EPA on bacterial diets within our 6 day lasting growth experiments.
However, it remains to be tested whether a potential limitation by EPA can be detected when more than one reproduction cycle is considered, i.e. when potential PUFA reserves are exhausted (cf. Martin-Creuzburg et al., 2009). Interestingly, EPA supplementation of the c-proteobacteria increased the survival of D. magna, suggesting that the detrimental effects mediated by these bacteria may have been alleviated by dietary EPA. We conclude that further (long-term) supplementation experiments are required before we are able to assess the role of PUFAs in determining the food quality of heterotrophic bacteria forDaphnia.
In the field, Daphnia do not feed solely on heterotrophic bacteria, and thus it is important to investigate at which dietary proportions bacteria become detrimental. By feeding D. magna with different combination of bacteria and the green alga S. obliquus, we show here that growth and egg production ofD. magnaare constraint when 80 20%, respectively, of the available carbon are represented by bacteria. This corroborates previous findings by Wenzel et al. (2012), who tested the food quality of a Pseudomonas strain in different combinations with Rhodomonas. They found that a 20% share of Rhodomonasin the food allowed survival ofDaphnia and that a 50% share enabledDaphniato reproduce.
Taipale et al. (2012) reported that high proportions of a type one methanotroph provided in different combi- nations with Cryptomonasresulted in high reproduc- tion of Daphniaover 2 weeks. At limiting quantities of phytoplankton, MOB could even partially support Daphnia growth (Deines & Fink, 2011). Thus, it appears that the dietary proportions at which bacteria become detrimental for Daphnia strongly depend on the bacterial phylotype and presumably also on the biochemical composition of the predominant algae.
Our results show that low proportions of heterotro- phic bacteria in the food suspension can even increase somatic growth rates ofDaphnia, as compared to a pure S. obliquus diet, suggesting that bacteria can provide essential nutrients not available in the green alga.
Bacteria are often characterized by high P:C ratios and the bacteria in our experiment, especiallyFlavobacte- rium sp., had higher P:C ratios than S. obliquus (cf.
Vadstein,2000). However, the P:C-ratio ofS. obliquus was already far above limiting levels (cf. DeMott, 1998; Persson et al.,2011) and thus a limitation ofD.
magnaby phosphorus was rather unlikely in particular because daphnids seem to incorporate phosphorus from
bacteria and algae with similar efficiencies (Wenzel et al.,2012). Nevertheless, low proportions ofFlavo- bacterium sp. and E. coli increased somatic growth rates of D. magna, suggesting that other bacteria- derived nutrients were responsible for the observed upgrading of the S. obliquus diet. Vitamins, for instance, which can be produced by many bacteria, including members of the Flavobacteria and Entero- bacteriaceae (Donderski & Nowacka, 1992), are potentially important for Daphnia and it has been suggested already that vitamin addition can improve the food quality ofS. obliquusforDaphnia (D’Agos- tino & Provasoli,1970; Mehdipour et al., 2011). The role of vitamins in determining food quality for Daphniacertainly requires further research.
Bacteria can comprise the major fraction of suspended organic matter in particular in oligo- to mesotrophic lakes (cf. Simon et al., 1992), but are quantitatively important also in eutrophic waters. High or increasing bacteria:phytoplankton ratios may con- strain growth and reproduction ofDaphniaeven under field conditions. However, low proportions of algae may be enough to compensate for the nutritional deficiency of the bacteria and the bacteria algae mixtures may supportDaphnia at least for a defined period of time. Especially members of Bacteroidetes, as well as of Actinobacteria (Taipale et al.,2012), even sustain Daphnia growth at higher bacterial propor- tions. Both bacterial groups are often numerically dominant in freshwater habitats, persist over seasons and seem to play an important role in the degradation of complex organic matter (Eiler & Bertilsson,2007;
Newton et al., 2011; Parveen et al., 2011). Conse- quently, they potentially gain in importance for Daphnianutrition at the end or between phytoplank- ton blooms (i.e. at higher bacteria:phytoplankton proportions).
Overall, our study highlight that feeding on heterotrophic bacteria can be associated with multiple challengesDaphniahave to cope with. Bacteria per se had detrimental effects onDaphnia because of their nutritional restraints or potential toxicity. Depending on the bacterial phylotype, however,Daphniamay be able to grow and reproduce even at high dietary proportions of bacteria, i.e. when provided in combi- nation with eukaryotic phytoplankton. Moreover, low bacterial proportions may even upgrade the nutritional value of phytoplankton-dominated food. Thus, we propose that the relative contribution of bacteria and
phytoplankton to total dietary carbon as well as their phylogenetic composition will strongly affect growth and survival of Daphnia and potentially other filter- feeding zooplankton under field conditions.
Acknowledgment We thank A. Wiese for technical assistance.
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